The long-term objective of the work in this research laboratory is to better understand the relationship between protein structure and function, including as well the relationship of amino acid sequence to higher-order structure and the relationship between biochemical and physiological function. Altered proteins associated with disease states typically display new properties. A better understanding of how new functions are acquired may lead to insight into how they may eventually be controlled. Our general approach to studying protein structure and function is to use molecular genetics and/or biochemistry to obtain proteins or protein fragments, assess their functions in vivo and in vitro by biochemical and genetic assays, and determine their structures at the required resolution by various biophysical methods. In the present competitive renewal, we propose to continue our ongoing studies of folding, stability, and dynamics in three protein systems, E. coli tr repressor (TrpR) E. coli arg repressor (ArgR), and yeast iso-1- cytochrome c(cytc). We plan to test whether the ordered self-assembly of TrpR proteolytic fragments is a good model for the folding pathway of intact TrpR, by using manual-mixing and/or stopped-flow CD, UV, and fluorescence spectroscopies to compare the kinetics of fragment assembly with the folding kinetics of intact TrpR. To examine the coupling between structural dynamics, thermal stability, and function of TrpR, we will conduct calorimetry and NMR experiments on the pure protein isolate from a temperature-sensitive TrpR mutant. We will test the role of a structurally important residue in TrpR dimer self-association by randomizing its codon, determining the phenotypes of each mutant in vivo and examining the purified mutant proteins by crosslinking, gel filtration, and fluorescence anisotropy. To examine the independence of protein domains in folding and stability, we will compare the structure, stability, multimeric assembly state, and folding mechanism of a chimeric TrpR derivative with those of wildtype TrpR by using biochemical methods and optical spectroscopies. We will test a preliminary model for domain organization of ArgR by purifying an N- terminal fragment and comparing its biochemical properties with those of intact ArgR. We plan to prepare isotopically-labelled ArgR fragment and attempt an NMR structure determination of the domain. The sequence and structural requirements for a helix-pairing reaction in the first step of the yeast iso-1-cytc folding pathway will be examined by making site- directed mutations, purifying and dissecting the proteins, and analyzing their fragment association reactions by CD, UV, fluorescence, calorimetry, and NMR.